Stroke and acute head trauma, multiple sclerosis and spinal cord injury are diseases for which so far there is no therapy available. Stroke is the third leading cause of death and the burden for the patients as well as for the social systems are enormous. In case of ischaemic stroke, which accounts for most of the strokes, blood vessel blockade in the brain is an initial event. Head trauma accidents are the leading disease of young people in the western world. A much smaller number of patients is affected by haemorhagic stroke caused by mechanical impact and artery rupture. It is a common feature, that approved pharmaceuticals are hardly available. Currently available treatment approaches are based upon pathophysiologic concepts derived from experimental work with focal cerebral ischaemia. These include pharmacological strategies for arterial recanalisation, inhibition of inflammatory processes and neural protection. Further work with arterial reperfusion strategies is under way. Early clinical studies with polymorphonuclear leukocyte-dependent endothelial adhesion receptor antagonists are being completed, but a strategy has yet to emerge. However any strategy addressing only a single step in the ischaemic cascade is likely to produce a modest benefit only. Therefor future therapies most properly will be based on combination therapies. A combination of low-dose acetylsalicylic acid (ASA) and modified-release dipyridamole has been shown to be additive in the secondary prevention of stroke (Tijssen et al., Int.J.Clin. Pract., suppl. 91, 14–16, 1997). Another combination which is currently proposed for clinical evaluation is tPA plus an effective neuroprotective agent. However the results of these studies are fare from being highly effective. Hence, there is urgent need to learn more about the pathophysiological mechanisms in order to provide drug targets that could be used to develop new drugs and establish more generally useful regimens for the treatment of patients suffering from acute neurodamaging effects and disorders such as multiple sclerosis.
This invention does focus on Ca2+-binding proteins since there has been considerable evidence for a role of Ca2+-binding proteins in neuroprotection. Although the precise function of calcium binding proteins (CaBPs) is not definitively known, it has been proposed that CaBPs act to buffer intracellular Ca2+ levels. Since Ca2+ overload activates biochemical processes leading to proteolysis and mitochondrial malfunction, this buffering capacity of CaBPs may have a protective effect against excitotoxic neuronal injury (Heizmann et al., TINS, 15, 259–64, 1992). A variety of evidences exists to support this proposed role of CaBPs in modulation of Ca2+ levels and in neuroprotection.
The major Ca2+-binding proteins (CaBPs) expressed in the central nervous system (parvalbumin, calbindin-D28K, and calretinin) have a very unusual and selective pattern of expression in various neuronal populations. Among neurons that do express CaBPs, most express only one type, although a small number of neurons express more than one of the major calcium binding proteins. There is growing evidence that the presence or absence of CaBPs in particular cell types underlies the phenomenon known as selective vulnerability. Selective vulnerability is a property of specific types of neurons to die in response to particular types of central nervous system (CNS) injury. For example, CA1 hippocampal neurons are selectively vulnerable to global ischemia, cerebellar Purkinje cells are selectively vulnerable to head trauma, stroke, and fetal alcohol exposure, and neurons in the substantia nigra are selectively vulnerable in Parkinson's Disease. An effort has been made to link selective neuronal vulnerability to the expression patterns of various CaBPs and some authors report that high levels of CaBPs are found in neuronal populations that are selectively vulnerable to injury, while others report high CaBP levels in neuronal populations that are selectively resistant to injury. For example, neurons expressing high levels of parvalbumin are reported to be selectively vulnerable to AMPA-induced toxicity (Weiss et al., Neurol., 40, 1288–1292, 1990), whereas cultured hippocampal neurons expressing high levels of calbindin-D28K are reported to be selectively resistant to glutamate-induced toxicity (Baimbridge et al., TINS, 15, 303–8, 1992). Similarly, hippocampal neurons expressing high levels of calretinin are resistant to toxic doses of the excitotoxins glutamate, NMDA, kainate, and quisqualate (Winsky et al., in: Novel Calcium-Binding Proteins, 277–300, 1991).
CaBPs have also been shown to have altered expression in various CNS disease states, but again the results are inconsistent about whether CaBP expression is related to selective vulnerability or selective resistance to injury. Neurons expressing calbindin-D28K are reported to be selectively vulnerable in Alzheimer's Disease (lacopino et al. PNAS, 87, 4078–82, 1990, Hof et al. Exp. Neurol., 111, 293–301, 1991) and Huntington's Disease (Kiyama et al., Brain Res., 526, 303–07, 1990), although calbindin-D28K-expressing neurons in the substantia nigra are not selectively vulnerable in Parkinson's Disease (Yamada et al., Brain Res., 526, 303–07, 1990). In a gerbil model of global ischemia, the presence of parvalbumin in certain hippocampal cell types has been shown to be positively associated with survival (Tortosa et al., Neurosci., 1, 33–43, 1993), although another study suggested that parvalbumin-expressing hippocampal interneurons are selectively vulnerable in Alzheimer's Disease (Brady et al., Neurosci., 80, 1113–25, 1997).
Mice with a knockout of the calbindin gene show functional deficits (e.g. ataxia) that suggest severe dysfunction in neurons normally expressing this CaBP (e.g., cerebellar Purkinje cells) despite the fact that these neurons appear morphologically normal. This finding suggests that CaBPs are vital to cellular activity patterns (Airaksinen et al., PNAS, 94, 1488–93, 1997). In addition, retroviral infection of motoneurons with calbindin-D28k has been shown to have neuroprotective effects against toxicity induced by IgG from patients with amyotrophic lateral sclerosis (Ho et al., PNAS, 93, 6796–801, 1996) and transfection with calbindin-D28k has been shown to protect PC12 cells from toxicity due to serum withdrawl, glutamate exposure, and the neurotoxin MPP+ (McMahon et al., Molec. Brain Res., 526, 303–07, 1998).
In conclusion, there is considerable information regarding a role for calcium binding proteins in neurodegeneration. It is certain that some CaBPs provide protection and others cause selective vulnerability, however it is not yet clear whether the expression of certain CaBPs within different neuronal populations results in different functional responses of a given CaBP. Another observation is that the severity of different types of CNS injury may affect the apparent neuroprotective efficacy of CaBPs—i.e., one CaBP may confer resistance in an injury model involving a mild injury, but may be unable to buffer Ca2+ increases in more severe CNS injuries. Thus there is considerable evidence that CaBPs confer resistance as well as vulnerability in CNS injury process, but the mechanism of this involvement and the regulation of the response has still to be worked out.
A gene familly comprising the functionally unidentified gene (MO25) was recently isolated from a mouse derived cDNA library (Miyamoto et al., Mol. Reprod. Dev., 34, 1–7, 1993). The library was constructed from RNA isolated from an early embrionic mouse. The predicted amino acid sequence for Mo25 revealed that the MO25 gene may have structural homology with Ca2+ binding proteins and lack membrane spanning domaines, indicating that the protein might be involved in cytosolic development of the unfertilized egg. However the real function of this protein remains unknown. Another Mo25 like gene, has been cloned from a Drosophila cDNA library (Nozaki et al. DNA Cell Biol., 15, 505–09, 1996). The deduced amino acid sequence of the Mo25 cDNA had 69.3% identity with mouse Mo25 homologe. A homologue in Saccharomyces cerevisiae encoded in an open reading frame near the calcineurin B subunit gene. Most recently another gene has been isolated from Aspergillus hym A mutants (Karos et al., Mol. Gen Genet., 260, 510–521, 1999) and turned out to correspond to the homologues in yeast, plants, fly, worm, fish, mice and man. A cellular function for the Hym protein has not yet been defined in any of the decribed organisms. As with many other other proteins where the functional contribution is only partially understood the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics”, that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superceding earlier approaches based on “positional cloning”. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
Functional genomics relies heavily on high-throughput DNA sequencing technologies and the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterise further genes and their related polypeptides/proteins, as targets for drug discovery. The discovery of a new human ANIC-BP-like protein splice variant and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of reproductive, immunological, vesicle trafficking, nervous system, developmental, and neoplastic disorders.